Reagent for determining the amount of cholesterol in serum and method of preparing same



Patented Sept. 26, 1961 REAGENT FOR DETERMINING THE AMOUNT OF CHOLESTERQL IN SERUM AND METHOD OF PREPARING SAME Quentin R. Hopper, 8849 Long Point, Houston 24, Tex. No Drawing. Filed Aug. 18, 1958, Ser. No. 755,395

6 Claims. (Cl. 252-408) This invention relates to cholesterol determination and more particularly to a reagent for use in such determination and a method of obtaining said reagent.

The amount of cholesterol in the blood stream isan indication of the general health of a patient. An excessive amount of cholesterol predisposes the patient to most all of the disesases relating to the circulatory system.

The amount of cholesterol in a sample of serum is normally determined by determining the density of cholesterol in a sample by use of a colorimeter. The colorimeter uses a filter according to the particular colorimeter employed which limits the color spectrum to a par ticular range. The sample is placed in the colorimeter and the density of the specimen determines the amount of light which passes through'and operates a bank of photoelectric cells in the colorimeter. These cells, in turn, operate a magnetic indicating device to indicate the density of the cholesterol in the sample. By use of conversion charts this density is transposed to a percentage factor of cholesterol in the sample.

A specimen of serum cannot be placed directly in the colorimeter but must be prepared in a special way. The prior art method requires approximately one hour. First the blood sample is combined with ether and alcohol and permitted to set for twenty or thirty minutes with periodic shaking. After the sample has set it is necessary to filter the sample to take out proteins which would render the solution cloudy. This filtering step takes approximately ten minutes. Thereafter, the sample solution is evaporated to the solid state by raising the temperature of the ether and alcohol to the boiling point. Obviously, this is a dangerous technique and laboratory technicians havebeen known to be severely burned in carrying out this method.

After the cholesterol solution has evaporated to the solid state, the residue is mixed with chloroform and the. chloroform solution is mixed with acetic anhydride and sulfuric acid. This mixture is placed in a dark room for approximately twenty minutes and then it is ready for reading in the colorimeter. In addition to the danger of carrying out this method, it is relatively expensive as a pure grade of ether must beused and the alcohol used is ethyl alcohol which requires the payment of alcohol taxes.

By this invention the time for preparing the blood serum for use has been reduced to approximately ten min.- utes and a sample may be prepared without danger to the technician. V o

It is an object of this invention to provide a reagent for determining the amount of cholesterol in a blood serum in which the specimen may be .prepared for use in a colorimeter much more rapidly than heretofore possible to thereby reduce the labor cost in cholesterol determination.

Another object is to provide areagent for use in preparing a blood stream for cholesterol determinationin a colorimeter in which the method of preparation does not require the periodic attention of a technician over a long period of time.

Another object is to provide a reagent which will reduce the time for determining the percentage of-cholesterol in serum from one hour to ten minutes.

Another object is to provide a reagent for use in cholesterol determination in which the ingredients are less expensive than those heretofore used.

Another object is to provide a method of preparation of a new reagent for cholesterol determination.

The reagent for use in cholesterol determination in accordance with this invention is a mixture of acetic anhydride, glacial acetic acid, and sulfuric acid. Preferably, the ingredients are. reagent grade, that is, refined for maximum concentration and purity. In the use of the re agent unhemolyzed blood serum is added to the reagent. As an example, 0.5 ml. of serum may be added to 11.5 ml. reagent within ten to fifteen seconds. Immediately thereafter, the sample may be read in a Leitz photrometer with filter 640. Of course, if a different photrometer is used a different filter may be required. The reading from the photrometer may be translated from the table in the handbook for the photrometer to give the concentration of cholesterol in mg. per ml. of serum.

The reagent mixture should, as noted above, be prepared from reagent grade constituents and each constituent should be present in approximately the percentages by volume given below:

Sulfuric acid between 4.5% and 22.5% Acetic anhydride not less than 40% Glacial acetic acid not less than 1% Preferably, the percentage by volume of the constituents is as follows:

Sulfuric acid, approximately 7.5% Acetic anhydride, approximately 55% Acetic acid, approximately 37.5%

In making up 100% mixtures, the minimum percentages of acetic anhydride and glacial acetic acid should be present. To make up the remainder of the solution it is preferred that either the glacial acetic acid or acetic anhydride be increased to provide a total of 100%. If desired other fillers may be used which do notimpair the mixture such at p-toluene sulfonic acid. For instance 1.7 ml. sulfuric acid may be added to 21 ml. of a mixture having 1 part glacial acetic acid, 5 parts 12% p-toluene sulfonic acid in a vehicle of glacial acetic acid, and 15 parts acetic anhydride; 7

Tests indicate that the percentages of sulfuric acid and the minimum percentages of glacial acetic acid and acetic anhydride are fairly critical. For instance, at approximately 4.5% and 22.5% sulfuric acid the results are marginal.

When less than about'40% of the acetic anhydride is present the necessary heat of reaction for the mixture is not present and the results are unsatisfactory.

When serum is added to pure acetic anhydride and sulfuric acid the result is very violent'agitation. This agitation is tamed by the addition of glacial acetic acid which should be present in at least 1% by volume which will tame the reaction sufiicient to avoid extreme danger. Also, the 1% glacial acetic acid tends to start the reaction in the desired direction.

It is believed that the chemical reactions are as follows: When the blood serum is added to the reagent the water in the blood causes the acetic anhydride to break into two molecules of glacial acetic acid. This produces the heat for carrying out the reaction. It is believed that when less than 40% by volume acetic anhydride is pres cut that insufiicient heat is developed for carrying out the reaction. As noted above, it is believed that the'glacial acetic acid tames the reaction by slowing it down and avoids the violent reaction present in the absence of acetic acid. The proteins in the serum are denatured by the acetic acid. Then the sulfuric acid breaks the denatured proteins into amino acids by removing water. The glacial acetic acid molecules then attach to the broken up amino acid molecules to prevent them from reattaching. This removes cloudiness which would be present if the proteins were not broken up in this manner. Also, molecules of acetic acid attach themselves to the cholesterol in the serum and in combining changes its chemical structure to bring it into a visible spectrum.

The sulfuric acid gives a strong acid medium which renders inert other constituents of the specimen and acts as a catalyst for the reaction. The water that is created by the activity of sulfuric acid on the proteins dilutes the sulfuric acid and gives the color of the specimen a little more stability.

It has been found that for some unknown reason the reagent deteriorates on about the eleventh day. It is believed that this deterioration is due to impurities in the reagent which could probably be neutralized to extend the life of the reagent. However, it has been found that no deterioration is present if the acetic anhydride and glacial acetic acid are first mixed and this mixture, together with the sulfuric acid retained in separate containers until just before cholesterol determination is to be made. Then, the sulfuric acid is added to the mixture of acetic anhydride and glacial acetic acid and no deterioration is present.

It is pointed out that acetic anhydride splits to form two molecules of glacial acetic acid in the presence of Water. Therefore, the glacial acetic acid could be provided by mixing water with acetic anhydride. This is, however, not preferred as impurities or other undesired reactions might result. Of course, this should be done in the absence of the sulfuric acid to insure the formation of the glacial acetic acid and to avoid diluting the sulfuric acid to a percentage solution which would not give the minimum amount of concentrated sulfuric acid necessary to carry out the reaction.

Given below in tabular form are experimental results of the use of the reagent of this invention in varying percentages of each of the constituents. The method used was to measure into a flask an amount of reagent grade acetic anhydride and glacial acetic acid. Thereafter, reagent sulfuric acid was added to the flask. Then, the proper quantity of centrifuged blood serum was gradually introduced and thereafter the specimen was placed in a colorimeter and the transmission of filtered light read on a calibrated instrument.

The following criteria were used in judging the results:

(a) The development quickly of a clear green solution, with no side reactions (such as yellow color or cloudiness).

(b) The absence of such great agitation as to make the procedure hazardous.

(c) The characteristic whereby the color intensity is relatively stable, so that the reading will not be dependent on the speed with which the technician reads his instrument.

Four observations were made for each experiment:

(1) The time necessary for the development of a clear green solution.

(2) The instrument reading immediately on development of said color.

(3) The rate of increase in the instrument reading.

(4) The lasting quality of the solution; that is, the rate of time for which the satisfactory green color persisted.

All concentrations which gave a reasonably satisfactory color and readings were considered satisfactory. Many of these did not give good results with regard to the rate of increase of the instrument reading and the lasting quality of the solution, but as they may beread immediately upon obtaining a satisfactory color, these two items were not considered to make the particular solutions unsatisfactory. The preferred reagent containing acetic acid 37.5%, acetic anhydride 55%, and sulfuric acid 7.5% gave satisfactory results on all counts.

It will be noted that experiments 1 through 12 were either marginal or satisfactory and that each of these ex- 4 periments utilize between 4.5% and 22.5 sulfuric acid, 40% or greater acetic anhydride and at least 1% glacial acetic acid. Experiments 13 through 19 fall without this range of compounds and were unsatisfactory.

Experiment No. Sulfuric Acetic Acetic Acid Acid Anhy- Results drtde 1 4. 5 0 95. 5 M 2 8 0 92 M 3. 22. 5 0 77. 5 M 4- 8 12 S 5. 9 26 65 S 6. 4. 5 30. 5 65 M 7 7. 5 37. 5 55 P 8. 9 41 50 S 9 15 35 50 S 10 8 52 40 M 11 14 46 40 M 12 8 46 46 S 13.. 0 35 65 U 14. 8 67 25 U 15. 7. 6 92. 4 0 U 16.. 22. 5 47. 5 30 U 17- 24 26 50 U 18.. 40 10 50 U 19.... 0 50 50 U Legend: U-unsatist'actory; M-marginal; S-satisfactory; P-preferred.

Experiments 1, 2 and 3 resulted in violent agitation. Addition of about 1% acetic acid has been found effective to tame the reaction and with this addition, experiment 2 becomes satisfactory. Experiments 1 and 3 remain marginal due to insuflicient and excessive sulfuric acid respectively.

From the above, it will be seen that between about 4.5% and 22.5% sulfuric acid is necessary as well as at least about 1% acetic acid and about 40% acetic anhydride. Of course, these are borderline percentages and it is preferred that the sulfuric acid be more than 4.5 but less than 22.5% and that more than 1% and 40%, respectively, of glacial acetic acid and acetic anhydride should be present.

In the use of the preferred reagent a mixture of 40.6% glacial acetic acid and 59.4% of acetic anhydride is prepared. 21 ml. of this mixture is placed in a flask, then 1.7 ml. of sulfuric acid is added. After mixing, the mix ture should be allowed to cool and should be kept in an amber bottle in a cool place. When desired to use, 11.5 ml. of the reagent is placed in a 125 ml. flask, then within ten to fifteen seconds add 0.5 ml. of unhemolyzed blood serummix and read immediately in a colorimeter. For instance, the reading may be done in a Leitz photrometer, model L.C.Z.F.L.-40 or a Leitz model L.C.Z.D.L.-22 using a 640 filter. These are both calibrated photrometers and each photrometer has a personal handbook correlated to the particular instrument. The reading from the particular photrometer should be referred to the table of the Leitz handbook accompanying the photrometer to determine the concentration of cholesterol in the mg. per ml. of serum.

The foregoing disclosure and description of the invention is illustrative and explanatory thereof and various changes in the size, shape and materials, as well as in the details of the illustrated construction, may be made within the scope of the appended claims without departing from the spirit of the invention.

What is claimed is:

1. A reagent for determining the amount of cholesterol in blood serum consisting essentially of, between approximately 4.5% and 22.5 reagent grade sulfuric acid by volume, at least approximately 1% reagent grade glacial acetic acid by volume, and at least approximately 40% reagent grade acetic anhydride by volume.

2. A reagent for determining the amount of the cholesterol in blood serum consisting of between approximately 4.5% and 22.5% reagent grade sulfuiic acid by volume,

With the exception of Nos. 1, 2, and 3 in the original experiments.

and suflicient reagent grade glacial acetic acid and acetic anhydride to provide a total of 100% by volume with a minimum volume of approximately 1% glacial acetic acid and approximately 40% acetic anhydride being present.

3. A reagent for determining the amount of cholesterol in blood serum consisting of approximately 7.5% reagent grade sulfuric acid by volume, approximately 37.5% glacial acetic acid by volume, and approximatelySS acetic anhydride by volume.

4. The method of formulating a reagent for determining the amount of cholesterol in serum comprising, mixing reagent grade acetic anhydride and reagent grade glacial acetic acid, and admixing said mixture withreagent grade sulfuric acid, said reagent, when mixed, consisting essentially of between approximately 4.5% and 22.5% sulfuric acid by volume, at least approximately 1% glacial acetic acid by volume, and at least 40% acetic anhydride by volume, said admixing of said mixture and sulfuric acid being carried out just prior to use of the reagent.

5. The method of formulating a reagent for determining the amount of cholesterol in serum comprising, mixing reagent grade acetic anhydride and reagent grade glacial acetic acid, and then admixing said mixture with reagent grade sulfuric acid, said reagent, when mixed, consisting of between approximately 4.5 and 22.5

sulfuric acid by volume, and sufficient glacial acetic acid and anhydride to provide a total of 100% by volume with 'a minimum volume of approximately 1% glacial acetic acid and approximately 40% acetic anhydride being present, said admixing of said mixture and sulfuric acid being carried out just prior to use of the reagent.

6. The method of formulating a reagent for determining the amount of cholesterol in serum comprising, mixing reagent grade acetic anhydride and reagent grade glacial acetic acid, and admixing said mixture with reagent grade sulfuric acid, said reagent, when mixed, consisting of approximately 7.5% by volume sulfuric acid, approximately 37.5 glacial acetic acid, and approximately acetic anhydride, said admixing of said mixture and sulfuric acid being carried out just prior to use of the reagent.

References Cited in the file of this patent Snell and Snell: Colorimetric Methods of Analysis, 3rd ed., vol. IV, Organic II, 1954, D. Van Nostrand Co. Inc., New York, page 372.

McDougal and Farmer: A Fluorometric Method for Total Serum Cholesterol, J. Lab. & Clin. Med, vol. 50, #3, September 1957, pages 485-487.

Allport et al.: Colorimetric Analysis, vol. 1, second ed., pub. 1957 by Chapman and Hall Ltd., of London, England, pages 85, 86, 87 and 88. 

1. A REAGENT FOR DETERMINING THE AMOUNT OF CHOLESTEROL IN BLOOD SERUM CONSISTING ESSENTIALLY OF, BETWEEN APPROXIMATELY 4.5% AND 22.5% REANGENT GRANDE SULFURIC ACID BY VOLUME, AT LEAST APPROXIMATELY 1% REAGENT GRADE GLACIAL ACETIC ACID BY VOLUME, AND AT LEAST APPROXIMATELY 40% REAGENT GRADE ACETIC ANHYDRIDE BY VOLUME. 